As membrane proteins exit the translocon, they complete the folding process in the membrane. Thus, it is important to determine how their sequences define their final structures and stabilities. This is generally approached by studying natural proteins. De novo protein design provides an alternative means to test and refine our understanding of membrane protein structure and function. By designing membrane proteins we can critically test and ultimately refine our understanding of recognition, folding and function. I Aim 1) we will define motifs that contribute largely to membrane protein folding and stability. We then determine the extent to which these motifs drive folding by designing peptides that idealize the sequence motifs. The association of peptides in phospholipid bilayers will be evaluated to determine the free energy of interaction, and how it changes upon changing residues thought to be key to the interaction. Aim 2) addresses the question of sequence-specific recognition in membranes. A variety of methods exist for the design or selection of antibodies and other reagents that recognize the water-soluble regions of proteins. However, companion methods for targeting Transmembrane (TM) regions are not generally available. Therefore, we developed a method for the computational design of peptides that targets TM helices in a sequence-specific manner. Now, we will use this computational procedure in conjunction with a bacterial screen of heterodimerization to better define the rules that govern the tight and specific association of helices. We will focus on the design of peptides that bind several biologically important systems including: EGF receptors (Collaboration with Natalia Jura), amyloid precursor protein (S. Prusiner), and the thrombopoietin receptor (Wei Tong). Aim 3) focuses on membrane metalloprotein assembly and metal ion transport. In the previous period, we designed model proteins that bind metal ion cofactors, including diiron and di-manganese centers in effort to determine how a protein matrix tunes the chemical properties of these cofactors to obtain diverse catalytic activities. Starting with proteins that bound metal ions but were catalytically inert we introduced substantial catalytic activities by varying the site's solvent accessibility, ligand identity, ligation geometry, and introducing substrate-binding sites. In Aim 3A, we now examine how immersion of the cofactor within a membrane will affect activity. In Aim 3B, we elaborate our design of TM metalloproteins to engineer channels that mediate passive transport down a concentration gradient as well as metal transporters that allow active transport against a concentration gradient. In preliminary results we have designed TM Zn(II) transporters that drive simultaneous proton export and Zn(II) import into liposomes. Here we characterize the conduction, structures, and dynamics (using solution and solids NMR and X-ray crystallography) of these designed transporters to determine the minimal features required for efficient facilitated transport of ions through TM helical bundles.